Human chondrosarcomas contain relatively high concentrations of proteoglycans formed by the non-covalent association of proteoglycan monomers, hyaluronic acid, and link protein. Link proteins are potentially valuable tumor marker proteins for human chondrosarcomas. Link protein was isolated from human chondrosarcomas by methods previously developed in our laboratory. Each of the human chondrosarcomas contained two link proteins with molecular weights of 44 and 48 kilodaltons.New methods involving lectin-agarose affinity chromatography were developed to isolate the chondrosarcoma link proteins to homogeneity. The relative binding affinity of link proteins to Ulex I, RCA I, RCA II, SBA, Con A, and WGA was determined. The link proteins bound to all lectins except Ulex I, and were eluted completed from RCA, SBA, and Con A with appropriate eluting sugars.However, no link protein could be eluted from WGA with GlcNAc. The 44 and 48 kilodalton link proteins have an unusually strong binding affinity for WGA. Using a linear gradient of 0 to 4 M GdmCl, 44 kilodalton link protein was eluted at low GdmCl concentrations and 48 kilodalton link protein was eluted at 4 M GdmCl. Lectin-agarose affinity chromatography is a valuable method for the separation of the two forms of link protein, and for the isolation of these two species to homogeneity. (1)